Cheaters shape the evolution of phenotypic heterogeneity in Bacillus subtilis biofilms

Abstract

Biofilms are closely packed cells held and shielded by extracellular matrix composed of structural proteins and exopolysaccharides (EPS). As matrix components are costly to produce and shared within the population, EPS-deficient cells can act as cheaters by gaining benefits from the cooperative nature of EPS producers. Remarkably, genetically programmed EPS producers can also exhibit phenotypic heterogeneity at single cell level. For instance, mature biofilms of Bacillus subtilis contain cells in an 'ON' state, expressing extracellular matrix genes, as well as cells in an 'OFF' state. Previous studies have shown that spatial structure of biofilms limits the spread of cheaters, but the long-term influence of cheating on biofilm evolution is not well understood. In addition, the influence of cheats on phenotypic heterogeneity pattern within matrix-producers, was never examined. Here, we examine the influence of EPS non-producers (cheaters) on phenotypic heterogeneity of matrix production within the populations of EPS producers (cooperators). We discovered that cheater-mediated evolution in pellicles leads to a transient shift in phenotypic heterogeneity pattern of cooperators, resulting in larger numbers of cells sustaining very high eps expression as depicted by hyper ON phenotype. Although, hyper ON strategy seems adaptive in presence of cheats, allowing favorable positioning of the WT in pellicles, it is soon substituted by hyper OFF phenotype and/or soon after by population collapse.This study provides additional insights on how biofilms adapt and respond to stress caused by exploitation in long-term scenario.

Evolved Biofilm: Review on the Experimental Evolution Studies of Bacillus subtilis Pellicles

“…The emergence of variants with lower or diminished matrix production in the B. subtilis biofilm raises the question of how such non-producers affect matrix production in the cooperative members of the population. Mutants deficient in production of EPS are not able to form robust pellicles alone, but can take advantage of the EPS-producing wild type, incorporate into the biofilm, and simultaneously save metabolic costs and exploiting a public good [34,39,61,62]. Experimental evolution has been performed on pellicles established by cooperative wild-type cells harboring a reporter for EPS production and non-producers that lack EPS production.…”

“…Experimental evolution has been performed on pellicles established by cooperative wild-type cells harboring a reporter for EPS production and non-producers that lack EPS production. These experiments revealed that in response to eps mutant incorporation into the B. subtilis pellicles, the wild type undergoes evolutionary adaptation by shifting the phenotypic heterogeneity of eps gene expression and becomes a matrix overproducer (hyper-ON), so that most of the cells express the eps genes [61]. At the same time, the fraction of eps mutants increases in the population that may result in the collapse of these populations where no pellicle is produced upon re-inoculation of the bacterial culture.…”